This project aims toward a better understanding of the structural and energetic basis of protein folding by elucidating the folding mechanisms for several small proteins with diverse structural characteristics. Mitochondrial and bacterial cytochromes c, staphylococcal nuclease, and peptide fragments are used as model systems. The structure, stability and dynamics of equilibrium states under native and denaturing conditions are investigated by 2D NMR and hydrogen-deuterium exchange methods. By coupling these approaches with quenched-flow techniques, a detailed characterization of H-bonded structure in transient folding intermediates is achieved. Together with optical studies, these pulsed NH exchange results provide a comprehensive description of the conformational transitions associated with folding and unfolding. Site-directed mutagenesis will be employed to evaluate the structural and energetic roles of individual side chains in stabilizing equilibrium and kinetic folding intermediates. The following objectives will be pursued: (l) early structural events in the folding of oxidized horse cytochrome c will be investigated by pulsed NH exchange under different refolding and labeling conditions; (2) equilibrium and kinetic analysis of mutants with changes at the interface between N and C terminal alpha-helices of cytochrome c and model peptide studies will provide insight into the structural and energetic basis for helix packing interactions and their kinetic role in folding; (3) the effects of mutations on the structure and dynamics of native and denatured cytochrome c will be characterized by NMR and hydrogen exchange methods, including a recently developed method that makes use of paramagnetic 1H NMR shifts as structural constraints; (4) the coupling of conformational transitions with ligand binding will be addressed by NMR, transient absorbance and photolysis studies on reduced cytochrome c; (5) comparison with parallel studies on the folding mechanism of the prokaryotic cytochrome c2 will provide insight into the evolution of folding pathways; (6) kinetic folding studies on staphylococcal nuclease and its extensive mutant library will be continued with emphasis on early structural events; (7) the conformation of peptides in their complex with antibodies and other large protein substrates will be investigated by quenched H-D exchange methods. Comparison of the folding mechanisms for model proteins with different structural characteristics will reveal some of the underlying general principles and test current theories of protein folding.